中国科学院昆明动物研究所机构知识库

转基因动物模型是一种病因学模型，能反映疾病的病理变化过程，被大量运用于基础研究、药物研发及临床测试等多个领域。特别在神经精神疾病领域，疾病具有较长的病程，常具有增龄性，随着患者年龄的增加而加重。然而传统基于化学诱导的动物模型仅能模拟疾病中晚期出现的临床症状，并不能反映疾病的发生发展过程，在神经精神疾病的早期诊断，机理研究及治疗等方面贡献较为有限。因此，转基因动物模型在此领域的研究工作中显得尤为重要。当前转基因动物模型主要集中在线虫、果蝇、斑马鱼及大小鼠等模式动物上，而人类神经精神疾病的复杂基因背景及症状往往很难在这些模式生物中得到客观体现，亟需发展一类转基因高等级动物的疾病模型。 非人灵长类动物与人类有十分相似的脑结构与功能系统，且与人类基因序列相似度高达93.54%。其中旧大陆猴，尤其是猕猴，最适宜用于研究神经系统疾病。国际上近年来对非人灵长类的基因操作技术获得了长足的进步，一系列基于胚胎转基因的猕猴动物模型相继构建成功，但因嵌合体问题不能有效解决，加之成模时间较长，猕猴仅能部分模拟疾病的病理表现却不能出现典型的临床症状，目前尚不能被广泛应用于基础研究及临床工作。基于此，我们计划制作基于特定脑区病毒注射转染的转基因猕猴模型，选用致病基因与发病脑区明确的阿尔兹海默病，帕金森氏综合征以及ReTT综合症作为建模候选疾病。此种方法建模具有成模时间短、操作简单易于大规模应用、转基因效率高以及可以在剔除其他脑区影响的背景下，探索单独脑区病变在整体疾病中的作用等优势。本实验首次采用多基因特定脑区转基因的猕猴建模。鉴于实验中涉及到很多无借鉴经验的关键技术与方法，我们首先在这些方面做了大量的建模前期奠基性工作：1.在载体病毒筛选方面，我们在4只猕猴及24只大鼠中比较了多种常用病毒载体在大鼠与猕猴脑内的表达成功率，发现多种病毒载体能够成功转染大鼠大脑但不能成功应用于猕猴，后续实验揭示提高腺相关病毒滴度后可以改善其在猕猴脑内的转染效果，但是此策略对慢病毒无效。同时在4只猕猴中比较了搭配不同启动子及血清型的腺相关病毒和慢病毒，实验结果显示2/9型腺相关病毒搭配多种启动子可以在猕猴脑内稳定表达。在此基础之上我们制作了过表达突变基因及基于CRISPR/Cas9的2/9型腺相关病毒用于建模。2.为了将病毒有效导入目标脑区，我们摸索了一套开骨瓣浅表脑区病毒转染及基于MRI指导下深部核团的病毒转染的手术范式。3.因猕猴资源宝贵，不能通过常规多时间点处死的方法来检测病毒在脑内的表达情况。为了更好的进行转基因建模，我们自主开发了一套基于光纤的病毒检测装置，能够于活体监测猕猴脑内病毒表达情况，用于在转基因建模的过程中实时监控病毒的表达情况。4.为了能更为客观的测试转基因猕猴的认知功能，我们还改进了一套基于电脑控制触屏自动训练及测试猕猴认知能力的行为学测试系统。在以上实验基础之上，1.我们初步建立了五只海马区敲除Methyl-CpG binding protein 2的ReTT综合症转基因猕猴模型，在注射病毒8周后该转基因猕猴开始出现昼夜节律异常，6个月后猕猴出现了自主活动减少，社交障碍等ReTT综合症的行为学改变。2.建立了4只过表达A30P基因的猕猴及8只敲除PINK及Parkin基因的帕金森综合症转基因猕猴，对转基因猕猴进行了最长一年的行为学检测。处死的8只建模猕猴均出现了典型的TH神经元死亡，α-synuclein聚集等PD病理症状；共同敲除PINK及Parkin基因组的猕猴出现了运动障碍，旋转改变等一系列类帕金森病行为学改变。3.最后我们成功制作了载荷量较大，较难保证病毒转染效率的阿尔兹海默病基因（APP, Ps1和Tau）的腺相关病毒，经测试以上病毒均能在猕猴脑内正常表达，其中APP及Ps1的病毒注射后，都能在短期内导致猕猴脑内Aβ沉积。

Transgenetic animal model is an etiology model, which can reflects the pathogenesis of disease progression and has hereditability. Therefore it has been widely used in basic research, drug development, clinical test, especially in the field of neurological disorders. At present, the transgenetic animal used in neuroscience are mainly concentrated on nematodes, fruit flies, zebrafish and rodents. However, the different genetic background between these animals and human are often make research on these models ineffective. Thus, there is an urgent need to develop new disease models with high-grade animals.Non-human primates are considered as ideal experimental animals for human disease modeling due to their high genetic similarity. Moreover, Non-human primates are similar to human in overall brain structure and social group living, thus providing a better reflection of the occurrence and development of human brain disorders. While, only few transgenic monkeys have been developed recently, and have not fully simulated the pathologies or symptoms of human disease, thereby have limited their widespread use.Therefore, we have planned to develop a new transgenetic monkey model by direct inject virus vector to transfection the specific brain regions of adult macaques. This transgenic technology has many advantages, such as simple operation, fast speed, high efficiency, which also allows us to exclude interferences from the other brain regions when observing the expression of pathogenic gene within the interesting brain regions. In the present study, we firstly used using the method of “virus injection” to build a series transgenetic monkey models, thus there were no relevant references to consult. Therefore, we have done a series of prerequisite works. (1) We have compared the transfection efficiencies of different viral vectors carrying different promoters and serotypes in monkeys, and found the optimal vectors to transfer exogenous genes into the central nervous system of monkey; (2) We have developed a system for detecting fluorescence signals in vivo, which could help us to estimate the degree of expression of the virus in transgenetic monkey; (3) We have improved the rhesus monkey touch screen cognitive test system.On this basis, we have chosen the disease with explicit pathogenic gene and lesion brain regions as the modeling disease, such as Alzheimer’s disease, Parkinson’s disease and Rett syndrome. Through inject the virus carrying pathogenic gene to disease related brain regions of macaques to build model, and assess the disease-like symptoms exhibited by the model animals to verify the availability of the model. In the present study, all three modeling disease models can occurred in a typical pathological feature of relevant disease. Moreover, the transgenetic Parkinson’s disease and Rett syndrome marquees were exhibiting some clinical symptoms.